Trypsin-like serine protease inhibitors, and their preparation and use

ABSTRACT

The invention relates to inhibitors of trypsin-like serine proteases of the general formula (I) which, as well as plasmin, also inhibit plasma kallikrein, and to their preparation and use as medicaments, preferably for treatment of blood loss, especially in the case of hyperfibrinolytic states, in organ transplants or heart surgery interventions, in particular with a cardiopulmonary bypass, or as a constituent of a fibrin adhesive.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/429,766, filed on Apr. 24, 2009, which is a continuation-in-part ofInternational Application No. PCT/EP2007/009220, filed Oct. 24, 2007,which claims benefit of German Patent Application No. 102006050672.3,filed Oct. 24, 2006. The disclosures of each application areincorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

The invention relates to inhibitors of trypsin-like serine proteases ofthe general formula

which, besides plasmin, also inhibit plasma kallikrein, and to thepreparation and use thereof as medicaments, preferably for the treatmentof blood loss, especially in hyperfibrinolytic conditions, in organtransplants or cardiac surgical procedures especially withcardiopulmonary bypass, or as constituent of a fibrin adhesive.

Inhibitors of plasmin and plasma kallikrein (PK) have been disclosed.Plasmin is a trypsin-like serine protease and cleaves numeroussubstrates C-terminally of the basic amino acids arginine or lysine.Plasmin is formed from the zymogen plasminogen by the catalytic actionof the plasminogen activators urokinase or tPA. Plasmin substratesinclude various proteins of the extracellular matrix and basal membrane,for example fibronectin, laminin, type IV collagen or fibrin, but alsonumerous zymogens such as proforms of the matrix metalloproteases or ofthe plasminogen activator urokinase. In blood, plasmin is responsible inparticular for fibrinolysis by cleaving fibrin into soluble products.

The endogenous plasmin inhibitors include α2-macroglobulin and theserpin α2-antiplasmin. Under certain pathological conditions there maybe spontaneous activation of fibrinolysis. In the event of such ahyperplasminemia, not only is the wound-closing fibrin degraded, butthere is also formation of anticoagulant fibrinogen degradationproducts. Serious impairments of hemostasis may arise thereby.Antifibrinolytics used clinically are synthetic amino carboxylic acidssuch as ε-aminocaproic acid, p-aminomethylbenzoic acid or tranexamicacid (trans-4-(aminomethyl)cyclo-hexanecarboxylic acid). These compoundsblock the binding of the zymogen plasminogen to fibrin and thus inhibitactivation thereof to plasmin. These compounds are therefore not directinhibitors of plasmin and are unable to inhibit plasmin which hasalready been formed. A further antifibrinolytic employed is aprotinin(Trasylol®, Bayer AG, Leverkusen), a polypeptide of 58 amino acids whichis obtained from bovine lung. Aprotinin inhibits plasmin with aninhibition constant of 1 nM, but is relatively nonspecific and alsoeffectively inhibits trypsin (K_(i)=0.1 nM) and plasma kallikrein(K_(i)=30 nM). Aprotinin also inhibits other enzymes, although withreduced activity.

A main use of aprotinin serves to reduce blood loss, especially incardiac surgical procedures with cardiopulmonary bypass (CPB), thusdistinctly reducing the need for perioperative blood transfusions (Sodhaet al., 2006). In addition, aprotinin is also employed in otheroperations, for example in organ transplants, to inhibit blood loss, oris used as addition in fibrin adhesives.

The use of aprotinin has several disadvantages. Since it is isolatedfrom bovine organs, there is in principle the risk of pathogeniccontamination and allergic reactions. The risk of an anaphylactic shockis relatively low with the first administration of aprotinin (<0.1%),but increases on repeated administration within 200 days to 4-5%.

It was recently reported that administration of aprotinin in directcomparison with ε-aminocaproic acid or tranexamic acid induces anincreased number of side effects (Màngano et al., 2006). Administrationof aprotinin led to a doubling of the number of cases of kidney damage,making dialysis necessary. Likewise, the risk of myocardial infarctionand apoplectic stroke was increased through administration of aprotininby comparison with the control groups.

To date only a few synthetic inhibitors of plasmin have been disclosed.Sanders and Seto (1999) described 4-heterocyclohexanone derivatives withrelatively weak activity, with inhibition constants of ≧50 μM forplasmin. Xue and Seto (2005) reported on peptidic cyclohexanonederivatives with IC₅₀ values of ≧2 μM, but further development thereofis unknown. Okada and Tsuda described various derivatives with a4-aminomethylcyclohexanoyl residue which inhibit plasmin with IC₅₀values of ≧0.1 μM, but clinical use of these inhibitors is not known(Okada et al., 2000; Tsuda et al., 2001).

Inhibition constants for plasmin have been published in numerouspublications on the development of inhibitors of coagulation proteasesas antithrombotics, where the aim in these cases was to inhibit plasminas weakly as possible. A possible use of these compounds for reducingblood loss in cardiac surgical procedures was not mentioned in any ofthese papers. Thus, for example, the thrombin inhibitor melagatraninhibits plasmin with a K_(i) value of 0.7 μM, whereas the structurallyclosely related compounds H317/86 has an inhibition constant of 0.22 μMfor plasmin (Gustafsson et al., 1998). However, both compounds inhibitthe protease thrombin distinctly more strongly with K_(i) values of ≦2nM, and thus administration of melagatran results in stronganticoagulation.

As described in the introduction, aprotinin inhibits not only plasminbut also plasma kallikrein (PK). PK is a multifunctional, trypsin-likeserine protease for which several physiological substrates are known.Thus, PK is able to release by proteolytic cleavage the vasoactivepeptide bradykinin from high molecular weight kininogen and to activatethe zymogens coagulation factor XII, pro-urokinase, plasminogen andpro-MMP 3. It is therefore assumed that the PK/kinin system has animportant role in various symptoms, for example in thromboembolicsituations, disseminated intravascular coagulation, septic shock,allergies, the postgastrectomy syndrome, arthritis and ARDS (adultrespiratory distress syndrome) (Tada et al., 2001).

Accordingly, aprotinin inhibits, by its inhibitory effect on PK, therelease of the peptide hormone bradykinin. Bradykinin has, viaactivation of the bradykinin B2 receptor, various effects. Thebradykinin-induced release of tPA, NO and prostacyclin from endothelialcells (see review paper by Schmaier, 2002) influences fibrinolysis,blood pressure and the inflammatory event. It is suggested that systemicinflammatory processes which may occur as side effect in operations arereduced by inhibiting bradykinin release.

Various bisbenzamidines such as pentamidine and related compounds, andesters of ω-amino- and ω-guanidinoalkylcarboxylic acids with micromolarvalues have been described as PK inhibitors (Asghar et al., 1976;Muramatu and Fuji, 1971; Muramatu and Fuji, 1972; Ohno et al., 1980;Muramatu et al., 1982; Satoh et al., 1985; Teno et al., 1991).

The first selective competitive inhibitors, which are derived fromarginine or phenylalanine, were developed by Okamoto et al., (1988) andinhibit PK with K_(i) values around 1 μM. Several papers on thedevelopment of competitive PK inhibitors have been published by theOkada group, with the most active compounds, which are derived fromtrans-4-aminomethylcyclohexanecarbonyl-Phe-4-carboxymethylanilide,having inhibition constants around 0.5 μM (Okada et al., 1999; Okada etal., 2000, Tsuda et al., 2001). It is common to the said PK inhibitorsthat they have a relatively high K_(i) value. U.S. Pat. No. 6,472,393described potent PK inhibitors with inhibition constants around 1 nM andhaving a 4-amidinoaniline as P1 residue. PK inhibitors have also beendescribed in U.S. Pat. No. 5,602,253. US 2006/0148901 described PKinhibitors whose inhibitory effect on plasmin is, however, relativelysmall, these inhibitors differing thereby from the inhibitors describedin the present application.

The invention is therefore based on the object of providing lowmolecular weight active substances which are suitable for therapeuticapplications and which reversibly and competitively inhibit inparticular plasmin and plasma kallikrein with high activity andspecificity and are therefore suitable for hemostasis in variousapplications, for example in cardiac surgical procedures with CPB, inorgan transplants or other operations. A further advantage of thesecompounds is that through their effect as inhibitor of plasma kallikreinin addition kinin release is reduced and thus kinin-mediatedinflammatory reactions can be suppressed. The kinin-induced release oftPA from endothelial cells is in turn suppressed by the inhibited kininrelease, it being possible thereby for fibrinolysis to be downregulatedby this mechanism. A further advantage of these compounds is, despiteselectivity, a certain inhibitory effect of these compounds on FXaand/or thrombin, and thus thrombotic complications are additionally tobe reduced on use of these compounds.

SUMMARY OF THE INVENTION

It has now surprisingly been found that it was possible to obtaininhibitors with strong inhibition constants for plasmin and plasmakallikrein by combining two sterically demanding and/or hydrophobicresidues R₂ and R₃ as shown in formula I, preferably substituted orunsubstituted aromatic systems. It was also possible to obtaincomparably good effects with substances having on R₂ nonaromatic and onR₃ basically substituted phenyl residues.

The present invention therefore relates to compounds of the generalformula (I)

-   -   with    -   R₁ optionally present one or more times and independently of one        another a COOR₅ residue, with R₅ equal to hydrogen or a branched        or linear lower alkyl group having 1-6 carbon atoms, preferably        methyl or ethyl, in particular methyl, a branched or linear        aminoalkyl residue having 1-6 carbon atoms, preferably methyl, a        halogen or pseudohalogen residue, preferably chlorine or a cyano        group, or a polyethylene glycol residue of the formula (II) or        (III)        CH₃—O—(CH₂—CH₂—O—)_(n)—CH₂—CH₂—(C═O)—NH—CH₂—  (II)        CH₃—O—(CH₂—CH₂—O—)_(n)—CH₂—CH₂—NH—(C═O)—CH₂—CH₂—(C═O)—NH—CH₂—  (III)    -    where n is ordinarily defined such that said polyethylene        glycol residues have an average molecular weight of 10 000 Da,        5000 Da, 3400 Da, 2000 Da, 1000 Da or 750 Da. Normally, n is an        integer between about 18 to about 250, in particular about 18,        about 25, about 50, about 85, about 125 or about 250.    -   R₂ an optionally substituted, aromatic or nonaromatic cyclic or        bicyclic system having 5-13 carbon atoms or aromatic heterocycle        having 4-5 carbon atoms and one nitrogen atom, nitrogen oxide,        oxygen atom or sulfur atom, especially a nitrogen atom or        nitrogen oxide; or a residue of the structure:

-   -   R₃ an optionally substituted, aromatic cyclic system having 5-6        carbon atoms or aromatic heterocycle having 3-5 carbon atoms and        1-2 nitrogen atoms, a nitrogen oxide, oxygen atom or sulfur        atom, especially a nitrogen atom or nitrogen oxide;    -   R₄ optionally a halogen residue which is present one or more        times, preferably fluorine;    -   o=1, 2 or 3, in particular 1;    -   p=0, 1, 2, 3 or 4, in particular 3; and    -   i=0 or 1, in particular 0;        and the racemic mixtures and salts with organic or inorganic        acids thereof.

Experimental results have shown that the inhibition of plasmin andplasma kallikrein is particularly good with compounds having cyclicstructures on R₂ and R₃ and in particular having an aromatic carbocyclicsystem on R₂ and R₃. It has further been possible to show that bysuitable choice of the substituents it is possible additionally toachieve a good inhibition of factor Xa and/or thrombin with compoundshaving an aromatic carbocyclic system on R₂ and R₃.

Experimental results have also shown that a marked reduction in theinhibition of thrombin is achieved when R₁ represents a 3-COOH group. Ina preferred embodiment, therefore, R₁ is present once and in meta orpara position, R₁ is preferably a COOH residue, and in particular R₁ ispresent once and is selected from hydrogen, a 4-COOH group or inparticular a 3-COOH group. It was possible to achieve a furtherreduction in the inhibition of thrombin by R₄ representing a fluorineatom, in particular in ortho position.

A further preferred embodiment of the present invention relates tocompounds in which R₂ is a substituted or unsubstituted, aromatic cyclicor bicyclic system having 6-13 carbon atoms or heterocycle having 5carbon atoms and one nitrogen atom.

The substitution on R₂ can be in general a halogen residue, preferablychlorine or fluorine, in particular chlorine, an optionallyfluorine-substituted, branched or linear alkyl residue having 1-6 carbonatoms, preferably methyl or tertiary butyl, an optionallyfluorine-substituted, branched or linear alkyloxy residue having 1-6carbon atoms, preferably methyl, a hydroxy residue or a cyano residue.

In an alternative embodiment, R₂ can also be a nonaromatic cyclic systemhaving 6 carbon atoms.

Particularly suitable compounds have proved to be compounds of theformula (I) in which the substitution on R₃ is an aromatic system withbasic residue, in particular an alkylamino residue having 1-3 carbonatoms, preferably 1 carbon atom, an amidino residue or guanidinoresidue. In particular, a compound of formula (I) with i=0 and withoutR₄ with the following residue has proved to be particularly suitable.

Com- pound No. R₁ R₂ p R₃ o 3 3- COOH

3

1

The salts of the compounds of the invention are generally formed fromhydrochloric acid, HBr, acetic acid, trifluoroacetic acid,toluenesulfonic acid or other suitable acids.

Compounds specifically suitable are those in which R₂ is selected fromthe following residues:

in particular

and/or in which R₃ is selected from in particular

Examples of such compounds are compounds of the formula (I) which aredefined as follows:

Compound No. R₁ R₂ p R₃ o i R₄ 1 H

3

1 0 — 2 4-COOH

3

1 0 — 3 3-COOH

3

1 0 — 4 3-COOH

3

1 1 — 5 3-COOH

3

1 0 2-F 6 H

3

1 0 — 7 H

3

1 0 — 8 H

3

1 0 — 9 H

3

1 0 — 10 H

3

1 0 — 11 H

3

1 0 — 12 H

3

1 0 — 13 H

1

1 0 — 14 H

1

1 0 — 15 H

1

1 0 — 16 4-COOH

1

1 0 — 17 H

1

1 0 — 18 3-COOH

1

1 0 — 19 4-COOH

1

1 0 — 20 H

1

1 0 — 21 H

3

1 0 — 22 H

0

1 0 — 23 3-COOH

0

1 0 — 24 H

2

1 0 — 25 3-COOH

2

1 0 — 26 H

2

2 0 — 27 H

2

2 0 — 28 H

3

1 0 — 29 4-COOH

3

1 0 — 30 3-COOH

3

1 0 — 31 H

3

2 0 — 32 H

3

1 0 — 33 3-COOH

3

1 0 — 34 H

3

2 0 — 35 4-COOH

3

2 0 — 36 3-COOH

3

2 0 — 37 H

1

2 0 — 38 H

1

2 0 — 39 H

3

2 0 — 40 3-COOH

3

2 0 — 41 H

3

2 0 — 42 3-COOH

3

2 0 — 43 H

1

2 0 — 44 H

2

2 0 — 45 H

1

2 0 — 46 H

0

2 0 — 47 H

2

2 0 — 48 H

2

3 0 — 49 H

3

2 0 — 50 3-COOH

3

2 0 — 51 H

3

2 0 — 52 3-COOH

3

2 0 — 53 H

3

2 0 — 54 3-COOH

3

2 0 — 55 H

3

2 0 — 56 3-COOH

3

2 0 —

It has also emerged that compounds of the general formula (IV)

which corresponds to the general formula (I), with

-   R₁, optionally present one or more times and independently of one    another a COOR₅ residue, with R₅ equal to hydrogen or a branched or    linear lower alkyl group having 1-6 carbon atoms, preferably methyl    or ethyl, in particular methyl, a branched or linear aminoalkyl    residue having 1-6 carbon atoms, preferably methyl, a halogen or    pseudohalogen residue, preferably chlorine or a cyano group, or a    polyethylene glycol residue of the formula (V) or (VI)    CH₃—O—(CH₂—CH₂—O—)_(n)—CH₂—CH₂—(C═O)—NH—CH₂—  (V)    CH₃—O—(CH₂—CH₂—O—)_(n)—CH₂—CH₂—NH—(C═O)—CH₂—CH₂—(C═O)—NH—CH₂—  (VI)    -   where n is defined such that the polyethylene chain has an        average molecular weight of 10 000 Da, 5000 Da, 3400 Da, 2000        Da, 1000 Da or 750 Da, n is preferably an integer between about        18 to about 250, in particular about 18, about 25, about 50,        about 85, about 125 or about 250;    -   R₂ a branched or linear alkyloxy residue having 1-6 carbon        atoms, preferably tertiary butyl, a hydroxyl residue, amino        residue or a branched or linear alkyloxycarbonylamido residue        having 1-6 carbon atoms, preferably tertiary butyl, or a        polyethylene glycol residue of the formula (V) or (VI) with n as        defined above;    -   R₃ selected from the following residues:

-   -   preferably

-   -   R₄ optionally a halogen residue which is present one or more        times, preferably fluorine,    -   o=1 or 2;    -   p=1, 2, 3 or 4, in particular 1 or 4,    -   i=0 or 1, in particular 0;        and the racemic mixtures and salts with organic or inorganic        acids thereof, are also suitable according to the present        invention.

Preferred compounds in this case also are those in which R₁ is presentonce and in meta or para position, R₁ is preferably hydrogen or a COOHresidue, and in particular R₁ is present once and is selected fromhydrogen, a 4-COOH group or a 3-COOH group.

The salts of these compounds are once again generally formed fromhydrochloric acid, HBr, acetic acid, trifluoroacetic acid,toluenesulfonic acid or other suitable acids.

Examples of such compounds are compounds of the formula (IV) with i=0and without R₄ residue, which are defined as follows:

Compound No. R₁ R₂ p R₃ o 57 H

4

1 58 4-COOH

4

1 59 H *—NH₂ 4

1 60 H

1

1 61 H *—NH₂ 1

1 62 H

4

2 63 H

1

1 64 4-COOMe

1

1 65 4-COOH

1

1 66 3-COOMe

1

1 67 3-COOH

1

1 68 H *—OH 1

1 69 4-COOMe *—OH 1

1 70 4-COOH *—OH 1

1 71 H

1

1 72 H *—OH 1

1 73 H

1

2 74 H *—OH 1

2 75 H

1

1 76 H *—OH 1

1 77 H

1

2 78 H *—OH 1

2

The compounds of the general formula I can be prepared in a manner knownin principle, as described hereinafter, for example as follows, with ingeneral the appropriate amino acids being coupled sequentially to anamidinobenzylamine protected at the amidino group. In this case, theN-terminal amino acid either already has the P4 residue, or the latteris subsequently linked thereto.

The nomenclature of the individual constituents P1, P2, P3 and P4 of thecompounds of the invention is evident hereinafter (see also Schechterand Berger, 1967).

For example, the protected, preferably Boc-protected, amidinobenzylaminewhich is protected at the amidino group, in particular4-acetyloxamidinobenzylamine, is obtained from the commerciallyavailable 4-cyanobenzylamine (Showa Denko K.K., Japan) by processesknown to a person skilled in the art. Cleavage of the protective groupis followed by coupling of the further amino acids and of the P4 residueby standard coupling methods and protective groups, preferably with Bocas N-terminal protective group. The P3 amino acid can also be coupleddirectly as protected, preferably benzylsulfonyl-protected, amino acidalready having the R1 residue. The peptide analogs are assembledsequentially, starting from the acetyloxamidinobenzylamine. Most of theintermediates crystallize well and can thus be easily purified. Thefinal purification of the inhibitors takes place at the last stage,preferably by preparative, reversed-phase HPLC.

The invention therefore further relates to a process for preparing acompound of the invention, where the appropriate amino acids are coupledsequentially to an amidino- or guanidinobenzylamine protected at theamidino or guanidino group, for example to a4-acetyloxamidinobenzylamine or to a4-(benzyloxycarbonylamidino)benzylamine, with the N-terminal amino acideither already having the P4 residue, or the latter subsequently beinglinked thereto. After possible purification, the resulting compounds canoptionally be PEGylated.

An exemplary process for preparing the compounds of the inventionincludes the following steps:

-   (a) amidation of an appropriate Nα-protected amino acid with the    residue R₃ with an appropriate protected aminomethylbenzamidine or    -guanidine,-   (b) after cleavage of the Nα-protective group of the amino acid with    R₃ reaction of the resulting product with the appropriate    benzylsulfonylamino acid with the residues R₁ and R₂ and cleavage of    remaining protective groups to give the compound of the invention    and, after a possible purification,-   (c) the resulting compound is optionally PEGylated.

Further process details which are generally known to a person skilled inthe art, e.g. concerning the chosen protective groups or the PEGylation,can be found in the examples. A preferred protective group of the amidenitrogen is for example tert-butyloxycarbonyl (Boc). The startingcompounds are, for example, amino acid derivatives or PEG derivatives.The chemicals can generally be obtained by purchase. The PEGylation,i.e. the derivatization with polyethylene glycol, generally took placeeither via the P3 amino acid or via the P4 benzylsulfonyl residue withactivated PEG derivatives, e.g. with PEG activated asn-hydroxysuccinimide ester.

An advantageous property of the PEG-coupled compounds is theprolongation of the half-life of the inhibitors in the bloodcirculation. The following structure shows an example in which the PEGchain has been coupled via the P3 amino acid (D-Lys).

The following compound was obtained by using a succinyl linker:

In addition, the PEG chain was coupled to via a suitableP4-benzylsulfonyl residue in accordance with the general formuladepicted below, with the P4 residue having been modified in the para orortho position with an aminomethyl group.

The following compound was obtained using a succinyl linker:

However, other preparation processes which can be carried out in thesame way are also known to a person skilled in the art. The PEGylatedcompounds are generally mixtures of compounds with various degrees ofPEGylation, and the molecular weight of the PEG residues is normally inthe region of 750, 1000, 2000, 3400, 5000 or 10 000 Da. However, otherspecific polyethylene glycols with defined molecular weight can also beobtained by purchase.

The present invention also extends to a medicament comprising at leastone of the compounds of the invention, preferably for the treatment ofblood loss, in particular in hyperfibrinolytic conditions, in organtransplants or cardiac surgical procedures, in particular withcardiopulmonary bypass.

The present invention also includes a fibrin adhesive which comprises atleast one of the compounds of the invention, in which aprotinin isreplaced by a suitable inhibitor of the present invention.

Fibrin adhesives generally mean a physiological two-component adhesivewhich comprises as first component fibrinogen, factor XIII and aprotininor at least one of the compounds of the invention, and as secondcomponent thrombin and calcium chloride for factor XIII activation.

The present invention also relates to the use of at least one compoundof the invention for the manufacture of a medicament of the invention orof a fibrin adhesive of the invention by processes generally known to aperson skilled in the art, e.g. by mixing with suitable excipients oradditives.

The present invention also relates to the following aspects:

Compounds of the General Formula

-   i is 0 or 1, preferably 0-   R₄ is H or OH, preferably H-   A is selected from the following structures:

-   where A is preferably a phenyl residue,-   R₁ is H, COOH, COOR₅ (with R₅=methyl or ethyl), aminomethyl,    halogen, pseudohalogen, but preferably H and COOH and particularly    preferably COOH, because the carboxyl group prolongs the half-life    of the inhibitors in the circulation,-   R₃ is

-   R₂ is branched or unbranched alkyl having 3-12 C atoms, also    cycloalkyl-substituted, aryl or aralkyl having 6-14 C atoms,    heteroaryl or as heteroarylalkyl having 6-12 C atoms and 1-3    heteroatoms, also

-   R₆ is halogen or pseudohalogen with p=0, 1, 2,-   and the racemates, crystal forms and hydrates and salts with organic    and inorganic acids thereof.

Compounds of the following formula in which the individual positions aredesignated P4-P1:

-   with-   P4=benzylsulfonyl residue unmodified or substituted-   P3=hydrophobic amino acid in the D configuration,    D-phenylpropylglycine and further amino acids-   P2=basic-hydrophobic amino acids in the L configuration-   P1=4-amidinobenzylamide residue and related groups.

Compounds of the following structure which are coupled to PEG via P3 asfollows:

The designations R₁, R₂, R₃, R₄, A and i correspond to the definitionsindicated above. B corresponds to the PEG chain which is in the form ofthe methyl ether at the end. Y is a suitable linker for coupling the PEGto the P3 amino acid, for example a propionyl residue, and X is eitheran NH or an NH-alkyl or NH-aryl group. The coupling takes place in amanner known per se.

The compounds of the invention have for example a PEG chain which has anaverage molecular mass of 750 Da, 1000 Da, 2000 Da, 5000 Da, 10 000 Da,20 000 Da or is a specific PEG chain.

Use of the compounds of the invention for the manufacture of amedicament which is suitable for reducing blood loss inhyperfibrinolytic conditions, and use of the compounds of the inventionas means for preparing a fibrin adhesive.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1K show the inhibitory effects of Compound No. 3, aprotinin,and tranexamic acid on t-PA-induced lysis of whole blood clots.

FIGS. 2A and 2B show the concentration-response curves ofantifibrinolytic efficacy of Compound No. 3, aprotinin, and tranexamicacid in human whole blood and plasma.

FIG. 3 shows the influence of Compound No. 3 and aprotinin oncoagulation parameters in vitro.

FIGS. 4A-4F show the effects of Compound No. 3 and aprotinin on thrombingeneration in platelet-rich human plasma.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the present invention relates to compounds of the generalformula (I)

-   -   wherein    -   R₁ is optionally present one or more times and each R₁ is,        independently, hydrogen or COOR₅;    -   R₂ is an optionally substituted ring system wherein said ring        system is selected from (i) an aromatic or nonaromatic cyclic or        bicyclic system comprising 5-13 carbon atoms, (ii) an aromatic        heterocycle comprising 4-5 carbon atoms and one nitrogen atom,        nitrogen oxide, oxygen atom, or sulfur atom, and (iii) a residue        of the structure:

-   -   R₃ is an optionally substituted ring system wherein said ring        system is selected from (i) an aromatic cyclic system comprising        5-6 carbon atoms, and (ii) an aromatic heterocycle comprising        3-5 carbon atoms and 1-2 nitrogen atoms, a nitrogen oxide,        oxygen atom, or sulfur atom;    -   R₄ is optionally present one or more times and each R₄ is,        independently, hydrogen or a halogen;    -   R₅ is hydrogen, a branched or linear lower alkyl group        comprising 1-6 carbon atoms, a branched or linear aminoalkyl        residue comprising 1-6 carbon atoms, a halogen or pseudohalogen        residue, or a polyethylene glycol residue of the formula (II) or        (III):        CH₃—O—(CH₂—CH₂—O—)_(n)—CH₂—CH₂—(C═O)—NH—CH₂—  (II)        or        CH₃—O—(CH₂—CH₂—O—)_(n)—CH₂—CH₂—NH—(C═O)—CH₂—CH₂—(C═O)—NH—CH₂—  (III)    -   wherein said polyethylene glycol residue has a molecular weight        of from 750 Da to 10,000 Da, and n is an integer from 25 to 250;    -   o=1, 2 or 3;    -   p=0, 1, 2, 3 or 4;    -   i=0 or 1;    -   and salts thereof.

In some embodiments, R₅ is methyl, ethyl, a branched or linearaminoalkyl residue comprising a methyl group, chlorine, a cyano group,or a polyethylene glycol residue of the formula (II) or (III) in which nis 18, 25, 50, 85, 125, or 250; R₂ is an optionally substituted ringsystem wherein said ring system is an aromatic heterocycle comprising4-5 carbon atoms and one nitrogen atom or nitrogen oxide; R₃ is anoptionally substituted ring system wherein said ring system is anaromatic heterocycle comprising 4-5 carbon atoms and one nitrogen atomor nitrogen oxide; R₄ is fluorine; o is 1; p is 3; and i is 0.

In some embodiments, R₁ is a COOR₅ residue present once and in the metaor para position; R₂ is an optionally substituted ring system whereinsaid ring system is phenyl, napthyl, pyridinyl, or pyridinyl-N-oxidegroup; R₃ is phenyl or phenyl substituted with an alkylamino residuehaving 1-3 carbon atoms; R₄ is hydrogen; R₅ is hydrogen or a branched orlinear lower alkyl group having 1-6 carbon atoms; o is 1 or 2; and i is0.

In some embodiments, R₁ is COOR₅ present once and in the meta or paraposition.

In some embodiments, R₅ is hydrogen.

In some embodiments, R₂ is an aromatic cyclic or bicyclic systemcomprising 6-13 carbon atoms or a heterocycle comprising 5 carbon atomsand a nitrogen atom.

In some embodiments, R₂ is an optionally substituted ring systemcomprising a substituent selected from a halogen residue, an optionallyfluorine-substituted branched or linear alkyl residue having 1-6 carbonatoms, an optionally fluorine-substituted branched or linear alkyloxyresidue having 1-6 carbon atoms, a hydroxy residue, or a cyano residue.In further embodiments, the substituent is selected from chlorine,fluorine, methyl, tertiary butyl, and OCH₃.

In some embodiments, R₂ is a nonaromatic cyclic system comprising 6carbon atoms.

In some embodiments, R₃ is a basic residue.

In some embodiments, R₃ is an optionally substituted ring systemcomprising a substituent selected from an alkylamino residue having 1-3carbon atoms, an amidino residue, and guanidino residue. In otherembodiments, substituent is an alkylamino residue comprising 3 carbonatoms.

In other embodiments, the compound is a salt selected from chloride,bromide, acetate, trifluoroacetate, and toluenesulfonate.

In some embodiments, R₂ is selected from the following residues:

In some embodiments, R₂ is

In some embodiments, R₃ is selected from

In certainembodiments, R₃ is

In some embodiments, the compound, or a salt thereof, is any ofCompounds Nos. 1-56 as described herein. In some embodiments, thecompound is Compound No. 3 which has the following structure:

or a salt thereof.

In another aspect, the invention features compounds of the generalformula (IV)

which corresponds to the general formula (I), wherein

-   -   R₁ is optionally present one or more times and each R₁ is,        independently, hydrogen or COOR₅;    -   R₂ is a branched or linear alkyloxy residue comprising 1-6        carbon atoms, a branched or linear alkyloxycarbonylamido residue        comprising 1-6 carbon atoms, or a polyethylene glycol residue of        the formula (V) or (VI) with n as defined below;    -   R₃ selected from the following residues:

-   -   R₄ is optionally present one or more times and each R₄ is,        independently, hydrogen or a halogen;    -   R₅ is hydrogen, a branched or linear lower alkyl group        comprising 1-6 carbon atoms, a branched or linear aminoalkyl        residue comprising 1-6 carbon atoms, a halogen or pseudohalogen        residue, or a polyethylene glycol residue of the formula (V) or        (VI)        CH₃—O—(CH₂—CH₂—O—)_(n)—CH₂—CH₂—(C═O)—NH—CH₂—  (V)        CH₃—O—(CH₂—CH₂—O—)_(n)—CH₂—CH₂—NH—(C═O)—CH₂—CH₂—(C═O)—NH—CH₂—  (VI)    -    wherein said polyethylene glycol residue has a molecular weight        of from 750 Da to 10,000 Da and n is an integer from 25 to 250;    -   o=1 or 2;    -   p=1, 2, 3 or 4;    -   i=0 or 1, in particular 0;

and salts thereof.

In some embodiments, R₅ is methyl, ethyl, a branched or linearaminoalkyl residue comprising a methyl group, chlorine, a cyano group,or a polyethylene glycol residue of the formula (V) or (VI) in which nis 18, 25, 50, 85, 125, or 250; R₂ is a branched or linear alkyloxyresidue comprising a tertiary butyl group or a branched or linearalkyloxycarbonylamido residue having a tertiary butyl group; R₃ is

R₄ is fluorine; p is 1 or 4; and i is 0.

In some embodiments, R₁ is COOR₅ present once and in meta or paraposition. In some embodiments, R₅ is hydrogen (e.g., a 4-COOH group or a3-COOH group).

In some embodiments, the compound is a salt selected from chloride,bromide, acetate, trifluoroacetate, and toluenesulfonate.

In some embodiments, the compound, or salt thereof, is any of CompoundNos. 60-78 as described herein.

In another aspect, the invention relates to a medicament that includesthe compound of the invention (e.g., a compound of formula (I)), or asalt thereof, and can further comprise suitable excipients or additives.In further embodiments, the medicament is for the treatment of bloodloss. In some embodiments, the blood loss occurs in hyperfibrinolyticconditions, in organ transplants, or cardiac surgical procedures. Theinvention also features fibrin adhesive comprising the compound of theinvention (e.g., a compound of formula (I)), or a salt thereof.

The following examples are intended to explain the invention in detailwithout restricting it.

EXAMPLES

1. Analytical Methods

1.1 Analytical HPLC

A Shimadzu LC-10A HPLC system consisting of the subsystems CTO-10AScolumn oven, LC-10AD pumps (2×), DGU-14A degaser, SIL-10AD autoinjector,SCL-10A system controller, SPD-10A UV-Vis detector and a Phenomenex Luna5 μm C18(2) 100 Å, 250×4.6 mm column, was used for the analyticalreversed-phase HPLC, utilizing the relevant Shimadzu CLASS-VP software,Version 5.3. Detection took place at 220 nm. Water with 0.1% TFA (A) andacetonitrile with 0.1% TFA (B) served as eluents at a flow rate of 1ml/min and a linear gradient (1% B/min). Different starting conditionswere used for the analytical HPLC depending on the compound, which areindicated for the corresponding compounds.

A Phenomenex Jupiter 5 μm C18(2) 300 Å, 250×4.6 mm column was used foranalyzing all the polyethylene glycol-modified active substances.

1.2 Preparative HPLC

A Shimadzu HPLC system consisting of the subsystems LC-8A preparativepumps (2×), DGU-14A degaser, FRC-10A fraction collector, SCL-10A systemcontroller, SPD-10A UV-Vis detector and a Phenomenex Luna 5 μm C8(2) 100Å, 250×30.0 mm column was used for the preparative RP-HPLC, utilizingthe relevant Shimadzu CLASS-VP software, Version 5.3. Detection tookplace at 220 nm. Water with 0.1% TFA (A) and acetonitrile with 0.1% TFA(B) likewise served as eluents, at a flow rate of 10 or 20 ml/min and asuitable gradient.

1.3 Mass Spectroscopy

The mass spectra were recorded routinely on a Finnigan ESI-MS LCQ(Bremen, Germany). All the polyethylene glycol-coupled compounds wereanalyzed in a Bruker Maldi Ultraflex T of/T of instrument.

Abbreviations Used

-   ACN Acetonitrile-   4-Amba 4-Amidinobenzylamide-   Ame Aminomethyl-   Boc tert.-Butyloxycarbonyl-   BSA bovine serum albumin-   Bzl Benzyl-   Bzls Benzylsulfonyl-   DIEA Diisopropylethylamine-   DCM Dichloromethane-   DMF N,N-Dimethylformamide-   HBTU 2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium    hexafluorophosphate-   HPLC High performance liquid chromatography-   MS Mass spectroscopy-   ONHS N-Hydroxysuccinimide ester-   NMM N-Methylmorpholine-   PEG Polyethylene glycol-   Phe(3-Ame) 3-Aminomethylphenylalanine-   Ppg Phenylpropylglycine-   RT Room temperature-   tBu tert.-Butyl-   Tfa Trifluoroacetyl-   TFA Trifluoroacetic acid-   TEA Triethylamine-   TMS-Cl Trimethylsilyl chloride-   Me Methyl    2. Synthesis of the inhibitors

2.1 3-HOOC-Bzls-d-Ppg-Phe(3-Ame)-4-Amba×2 acetate (3)

a) Boc-Phe(3-Ame)-OH×acetate

5 g (17.2 mmol) of Boc-Phe(3-CN)—OH (Acros Organics) were dissolved in700 ml of 90% strength acetic acid and hydrogenated with hydrogen underatmospheric pressure and 800 mg of 10% Pd/C as catalyst at 40° C. for 3hours. The solvent was removed in vacuo, and the residue was dissolvedin a small amount of methanol and precipitated by adding diethyl ether.

Yield: 4.1 g (HPLC: 16.7 min, Start with 10% B)

b) Boc-Phe(3-Tfa-Ame)-OH

4.6 g (13 mmol) of Boc-Phe(3-Ame)-OH×acetate were dissolved in 30 ml ofmethanol, and 4 ml (29.9 mmol) of DIEA and 2 ml (16.78 mmol) of ethyltrifluoroacetate were added at room temperature. The mixture is stirreduntil the original suspension has completely dissolved after about 15min. After one hour, the solvent is removed in vacuo, and the residue isdissolved in ethyl acetate and water. The ethyl acetate phase is washed2× with 5% KHSO₄ solution and 3× with saturated NaCl solution, and theorganic phase is dried with Na₂SO₄. The solvent is removed in vacuo.

Yield: 4.9 g of amorphous solid (HPLC: 28.13 min, Start with 20% B)

c) Boc-Phe(3-Tfa-Ame)-4-(acetylhydroxyamidino)benzylamide

5.43 g (13.9 mmol) of Boc-Phe(3-Tfa-Ame)-OH and 4.28 g (15.3 mmol) of4-(acetylhydroxyamidino)benzylamine (synthesis described in thesupplement to Schweinitz et al., 2004) were dissolved in 50 ml of DMFand, at 0° C., 5.2 ml (30 mmol) of DIEA and 5.81 g (15.3 mmol) of HBTUwere added. The mixture is stirred at 0° C. for 15 min and at RT for afurther 3 h. The solvent is removed in vacuo, and the residue isdissolved in ethyl acetate. The ethyl acetate phase is washed 3× with 5%KHSO₄ solution, 1× with saturated NaCl solution, 3× with saturatedNaHCO₃ solution and 2× with saturated NaCl solution. The product whichprecipitates between the phases is filtered off with suction and driedin vacuo.

Yield: 4.17 g of white crystals (HPLC: 28.08 min, Start with 20% B)

d) H-Phe(3-Tfa-Ame)-4-(acetylhydroxyamidino)benzylamide×HCl

4.1 g of Boc-Phe(3-Tfa-Ame)-4-(acetylhydroxyamidino)benzylamide weresuspended in 60 ml of dry dioxane, and 11 ml of 4 N HCl in dioxane wereadded. After brief ultrasonic treatment, the mixture is shaken at roomtemperature for 1 h. After 1 h, the product is precipitated by addingdiethyl ether and is filtered off with suction and dried in vacuo.

Yield: 3.8 g of white solid (HPLC: 9.47 min, Start with 20% B)

e) 3-MeOOC-Bzl-SO₃ ⁻×Na⁺

5 g (21.8 mmol) of methyl 3-bromomethylbenzoate (Acros Organics) weresuspended in 25 ml of water, and 2.94 g (23.8 mmol) of Na₂SO₃ wereadded. The mixture was refluxed for 5 h and then part of the solvent wasremoved in vacuo until crystallization had started. The mixture wasstored at 4° C. overnight, and the product was filtered off.

Yield: 3.7 g of white crystals (HPLC: 12.02 min, Start with 10% B)

f) 3-MeOOC-Bzls-Cl

2.5 g (9.91 mmol) of 3-MeOOC-Bzl-SO₃ ⁻×Na⁺ were moistened withphosphoryl chloride, and 2.27 g (10.9 mmol) of PCl₅ were added. Themixture was cooled at 0° C. for about 5 min and then heated on an oilbath (bath temperature 80° C.) for 4 h. The mixture was then poured ontoice and vigorously stirred. After stirring for about 30 min, the acidchloride begins to precipitate and is filtered off with suction anddried in vacuo.

Yield: 1.4 g of white solid

g) 3-MeOOC-Bzls-d-Ppg-OH

1.3 g (6.72 mmol) of H-d-Ppg-OH (Peptech, Burlington, Mass.) weresuspended in 90 ml of dry DCM, and 2 ml (15.7 mmol) of TMS-Cl and 2.6 ml(15 mmol) of DIEA were added. The mixture was refluxed for 1 h, theclear solution was cooled to 0° C., and 2 g (8 mmol) of 3-MeOOC-Bzls-Cland 2.6 ml of DIEA were added. The mixture was stirred at 0° C. for 15min and at RT for 1.5 h. The solvent was removed in vacuo, and theresidue was dissolved in 700 ml of half-saturated NaHCO₃ solution. Themixture was extracted 2× with a little ethyl acetate, and then theaqueous phase was acidified with HCl (pH about 2-3). The mixture isextracted 3× with 150 ml of ethyl acetate, and the combined ethylacetate phase is washed 2× with 5% KHSO₄ solution and 1× with saturatedNaCl solution. The organic phase is dried with Na₂SO₄, and the solventis removed in vacuo.

Yield: 2.4 g of oil (HPLC: 33.53 min, Start with 20% B)

h) 3-MeOOC-Bzls-d-Ppg-Phe(3-Tfa-Ame)-4-(acetylhydroxyamidino)benzylamide

0.605 g (1.5 mmol) of 3-MeOOC-Bzls-d-Ppg-OH and 0.85 g (1.65 mmol) ofH-Phe(3-Tfa-Ame)-4-(acetylhydroxyamidino)benzylamide×HCl were dissolvedin 40 ml of dry DMF and, at 0° C., 0.63 g (1.65 mmol) of HBTU and 0.6 ml(0.34 mmol) of DIEA were added. The mixture is stirred at 0° C. for 15min and at RT for a further 3 h. The solvent is removed in vacuo, andthe residue is dissolved in ethyl acetate. The ethyl acetate phase iswashed 3× with 5% KHSO₄ solution, 1× with saturated NaCl solution, 3×with saturated NaHCO₃ solution and 2× with saturated NaCl solution. Thesolvent is removed in vacuo.

Yield: 1.36 g of oil (HPLC: 38.40 min, Start with 20% B)

i) 3-MeOOC-Bzls-d-Ppg-Phe(3-Tfa-Ame)-4-amidinobenzylamide×acetate

1.3 g of 3-MeOOC-Bzls-d-Ppg-Phe(3-Tfa-Ame)-4-(acetylhydroxyamidino)benzylamide are dissolved in 100 ml of 90% acetic acid and hydrogenatedwith hydrogen under atmospheric pressure and 150 mg of 10% Pd/C ascatalyst overnight. The catalyst is filtered off and the filtrate isconcentrated in vacuo.

Yield: 1.2 g of oil (HPLC: 29.45 min, Start with 20% B)

2.2 3-HOOC-Bzls-d-Ppg-Phe(3-Ame)-4-amidinobenzylamide×2 acetate (3)

1.2 g of 3-MeOOC-Bzls-d-Ppg-Phe(3-Tfa-Ame)-4-amidinobenzylamide×acetatewere stirred in a mixture of 10 ml of dioxane and 10 ml of 1 N LiOH for1.5 h. The mixture was then neutralized by adding TFA, and the productwas purified by preparative reversed-phase HPLC. The product-containingfractions were combined and lyophilized.

Yield: 0.4 g as TFA salt (HPLC: 24.16 min, Start with 10% B)

MS: calculated: 698.29 found: 699.3 (M+H)⁺

The product was converted into the acetate salt by preparative HPLC byelution with an increasing acetonitrile gradient containing 0.1% aceticacid.

Yield: 0.32 g

Further inhibitors were synthesized in accordance with the abovesynthesis description, incorporating differently substituted orunsubstituted benzylsulfonyl residues and various P3 amino acids asreplacement for d-phenylpropylglycine. Further analogs ofd-phenylpropylglycine were synthesized by Heck coupling and incorporatedinto the P3 position of the inhibitors. The synthesis can be carried outfor example as follows:

2.3 Bzls-d-Gly(3-O-Phpr)-Phe(3-Ame)-4-Amba×2 TFA (6)

a) Bzls-d-Gly(allyl)-OH

1.0 g (8.68 mmol) of D-allylglycine (Peptech, Burlingtom, Mass.) wassuspended in 50 ml of dry DCM, and 2.4 ml (19 mmol) of TMS-Cl and 3.3 ml(19 mmol) of DIEA were added. The mixture was refluxed for 1 h, theclear solution was cooled to 0° C., and 2.35 g (9.55 mmol) of Bzls-Cland 1.8 ml of DIEA were added. The mixture was stirred at 0° C. for 15min and at RT for 1.5 h. The solvent was removed in vacuo and theresidue was dissolved in 700 ml of half-saturated NaHCO₃ solution. Themixture was extracted 2× with ethyl acetate and then the aqueous phasewas acidified with HCl (pH about 2-3). The mixture was extracted 3× with150 ml of ethyl acetate, and the combined ethyl acetate phase was washed2× with 5% KHSO₄ solution and 1× with saturated NaCl solution. Theorganic phase was dried with Na₂SO₄, and the solvent was removed invacuo.

Yield: 2.2 g of oil (HPLC: 21.1 min, Start with 20% B)

MS (ESI, m/e): 267 [M−1]⁻

b) Bzls-d-Ala(3-Cl-styryl)-OH

A suspension of 0.476 g (1.48 mmol) of tetra-n-butylammonium bromide,0.34 g (4.05 mmol) of NaHCO₃, 0.32 g (1.34 mmol) of 1-Cl-iodobenzene and9 mg (0.04 mmol) of palladium(II) acetate in a mixture of 2.5 ml of DMFand 2.5 ml of water is stirred at RT for 10 min. A solution of 0.4 g(1.48 mmol) of Bzls-d-Gly(allyl)-OH in 2.5 ml of DMF and 2.5 ml of wateris added to this suspension, and the mixture is heated at 45-50° C. for4-6 days, repeatedly supplementing where appropriate with small amountsof catalyst. The catalyst is filtered off, the solvent is removed invacuo, and the residue is suspended in 50 ml of 5% KHSO₄ solution. Themixture is extracted 3× with 15-20 ml of ethyl acetate each time, andthe combined ethyl acetate phase is washed 2× with 5% KHSO₄ solution and1× with saturated NaCl solution. The organic phase is dried with Na₂SO₄,and the solvent is removed in vacuo. The residue (0.55 g of dark oil) ispurified by flash chromatography on silica gel 60 (40-63 μm) (gradient0-20% methanol in DCM).

Yield: 0.23 g (HPLC: 39.7 min, Start with 20% B)

Further assembling of the inhibitor took place in analogy to thesynthesis described for inhibitor 1. The intermediate 2.2.b was coupledto the intermediate 2d(H-Phe(3-Tfa-Ame)-4-(acetylhydroxyamidino)benzylamide×HCl) in analogy tomethod 2h. The resulting intermediate was hydrogenated in analogy tomethod 2i, but in this case no cleavage of the Cl atom of the P3 aminoacid was observed. In the last step, the trifluoroacetyl protectivegroup was cleaved off with LiOH in dioxane in analogy to the finalsynthesis step in the preparation of compound 3.

HPLC: 29.04 min, Start with 10% B

MS: calculated: 688.26 found: 689.2 (M+H)⁺

2.4 3-HOOC-Bzls-d-Ppg-Phe(3-Ame)-4-guanidinobenzylamide×2 acetate (4)

Compound 4 was also synthesized in analogy to the above synthesisdescription 2.2a-i, using p-nitrobenzylamide for step c) instead of4-(acetylhydroxyamidino)benzylamide. The nitrobenzylamide residue wasreduced to the p-aminobenzylamide in analogy to step 2.2i withmethanol/THF (1:1) as solvent. The guanylation of the p-aminobenzylamideresidue took place with commercially available1,3-di-Boc-2-(trifluoromethylsulfonyl)guanidine (Fluka) as guanylatingreagent. For this purpose, the intermediate from the reduction wasdissolved in dioxane and stirred with the guanylating reagent and TEA at50° C. for 1 day. After the solvent has been evaporated, the Bocprotective groups were cleaved off with TFA in a known manner. After thesolvent had been evaporated, the trifluoroacetyl protective group andthe methyl ester were cleaved off in the last step using LiOH in dioxanein analogy to the final synthesis step in the preparation of compound 3.

HPLC: 25.1 min, Start with 10% B

MS: calculated: 713.3 found: 714.4 (M+H)⁺

2.5 Pegylated Compounds

Further inhibitors to which polyethylene glycol (PEG) chains of varyingchain length were covalently coupled were synthesized by standardprocesses. Commercially available PEG derivatives from Fluka, NektarTherapeutics or Rapp Polymere with different average molecular weights(1000 Da, 2000 Da, 5000 Da, 10 000 Da) were used for all the syntheses.The PEG derivatives used are protected as methyl ether at one end andmodified at the other end with a propionic acid or succinic acid residueactivated as N-hydroxysuccinimide ester. It was thus possible to reactthese activated PEG derivatives with a free amino group of the inhibitor(see synthesis schemes 1 to 5). In the last step, the TFA protectivegroup was cleaved off by mixing with 1 N NaOH solution, and the productswere purified by ion exchange chromatography on Fractogel® CE (MerckKGaA, Darmstadt) using an ammonium acetate gradient and were lyophilized3× from water. The following examples yielded inhibitors with a PEGchain of an average mass of about 1000, 2000, 5000 or 10 000 Da.

The compounds synthesized in the examples are summarized, includingtheir inhibition constants, in the table below.

3. Determination of the Inhibition Constants for Plasmin and PK (K_(i)Values in nM)

The inhibitory effect for the individual enzymes was determined inanalogy to a previously disclosed method (Stürzebecher et al., 1997).

The reactions to determine the inhibition of human plasmin and humanplasma kallikrein were carried out in the following mixture at 25° C.:

-   -   200 μl of TBS (0.05 M trishydroxymethylaminomethane; 0.154 M        NaCl, 2% ethanol, pH 8.0; contains the inhibitor)    -   25 μl of substrate (2 mM, 1 mM and 0.67 mM        tosyl-Gly-Pro-Lys-pNA=Chromozym PL from LOXO for plasmin and 2        mM, 1 mM and 0.5 mM H-D-Pro-Phe-Arg-pNA=S2302 from Chromogenix        for PK, dissolved in H₂O)    -   50 μl of enzyme solution (plasmin from Calbiochem: 2-5 mU/ml in        0.154 M NaCl+0.1% BSA m/v+25% v/v glycerol; plasma kallikrein        from Enzyme Research Lab.: 20-60 ng/ml in 0.154 M NaCl+0.1% BSA        m/v)

For zero order kinetics, the reaction was stopped after 20 min by adding25 μl of acetic acid (50% v/v), and the absorption at 405 nm wasdetermined using a Microplate Reader (Multiscan Ascent, from Thermo). Inthe case of pseudo-first order kinetics, the reaction rates in theequilibrium state were ascertained by recording the reaction kinetics.The K_(i) values were ascertained either in accordance with Dixon (1953)by linear regression using a computer program or by parameter fitting inaccordance with the rate equation for competitive inhibition. The K_(i)values are the average of at least two determinations.

TABLE K_(i) values: A means <10 nM, B means <100 nM, C means <1000 nMand D means >1000 nM 1^(st) group Mass HPLC Plasmin PK Xa Thrombin No.(found/calculated) % AN [K_(i)] [K_(i)] [K_(i)] [K_(i)] 1 655.3/654.337.4 A A B C 2 699.5/698.3 33.6 A A B C 3 699.3/698.3 34.1 A A B D 4714.4/713.3 35.1 B A D D 5 717.3/716.3 34.9 A A B D 6 689.2/688.3 39.8 AA B C 7 689.3/688.26 41.9 A A A B 8 689.3/688.26 39.0 A A B B 9669.3/668.3 39.3 A A B C 10 685.6/684.3 37.2 A A B C 11 723.2/722.3 41.6A A A C 12 739.4/738.3 44.4 A A B C 13 683.3/682.3 41.9 B A B C 14703.3/702.3 40.7 A A B C 15 677.3/676.3 37.4 A A B B 16 721.2/720.3 34.3A A C C 17 677.3/676.3 37.7 A A B C 18 721.1/720.3 35.6 A A B D 19721.2/720.3 34.6 A A C C 20 633.8/632.3 36.1 A A B C 21 661.4/660.3 41.5A A B C 22 703.4/702.3 38.4 A A B A 23 747.3/746.3 36.3 A A B B 24657.3/656.3 29.1 A A A C 25 747.3/746.3 36.3 A A B D 26 671.4/670.3 30.7A A A C 27 642.3/641.3 42.4 A A A C 28 655.4/654.3 37.0 A A C C 29699.4/698.3 31.8 A A C C 30 699.4/698.3 34.2 A A C D 31 669.4/668.3 36.9A B C B 32 616.2/615.3 35.3 A B C B 33 660.3/659.3 31.9 A B C D 34640.4/639.3 52.7 A A C B 35 684.3/683.3 48.6 A A C C 36 684.3/683.3 49.8A A B C 37 647.9/646.3 39.3 B B B B 38 660.6/659.4 39.3 A B B A 39641.5/640.3 36.1 A A B A 40 685.5/684.3 32.2 A A C B 41 657.5/656.3 40.0A A C B 42 701.5/700.3 35.4 B A D C 43 619.2/618.3 35.1 A B B A 44627.2/626.3 33.7 B B B B 45 638.8/637.3 30.6 A A C B 46 638.7/638.2 34.0A A C B 47 643.3/642.3 28.0 A A A A 48 641.4/640.3 34.4 A A A C 49641.4/640.3 33.7 A A B B 50 685.3/684.3 31.5 A A B C 51 641.3/640.3 33.9A A A A 52 685.2/684.3 31.5 A A A B 53 657.3/656.3 36.7 A A A B 54701.4/700.3 34.4 A A B D 55 657.4/656.3 37.1 A A A B 56 701.5/700.3 34.9A A A C 2^(nd) group: MS HPLC # (found/calculated) % AN Plasmin PK XaThrombin 57 708.6/707.38 36.5 A A B D 58 752.4/751.4 31.9 B A C D 59608.4/607.3 41.0 B A C D 60 666.8/665.3 34.4 B A C D 61 566.5/565.3 21.5B A D D 62 694.9/693.4 34.6 A A C C 63 623.6/622.3 34.8 A A C C 64681.7/680.3 34.7 A A D D 65 667.7/666.3 30.1 B A C D 66 681.3/680.8 34.4A A B D 67 667.5/666.8 30.1 A A B D 68 567.8/566.3 25.4 B A D D 69625.6/624.3 26.1 B A D D 70 611.9/610.3 21.0- B A D D 71 623.7/622.333.9 A A D C 72 567.7/566.3 24.6 B B D D 73 637.6/636.3 35.1 B B C B 74581.5/580.3 25.2 B B D D 75 636.5/635.3 33.2 B B C C 76 580.6/579.3 23.1B B D D 77 650.6/649.3 35.0 A A C B 78 594.6/593.3 25.1 B A D DPEGylated compounds: Mass HPLC Plasmin PK Xa Thrombin No.(found/calculated) % AN [K_(i)] [K_(i)] [K_(i)] [K_(i)] 79a  ~1000 Da34.9 A A C D 79b  ~2000 Da 39.4 A — — — 79c  ~5000 Da 43.6 A A B D 79d~10000 Da 45.4 A A C D 80 ~10000 Da 45.8 A A B D 81 ~10000 Da 46.4 A A CC 82 ~10000 Da 46.0 A A C C 83 ~10000 Da 45.2 A A C CResults:

-   1. The K_(i) value of the plasmin inhibition was generally <100 nM.    The K_(i) value was distinctly lower than 100 nM in particular for    compounds with cyclic structures at R₂ and R₃ and was below about 10    nM for compounds with an aromatic carbocyclic system on R₂ and R₃.    Surprisingly there is a particularly large number of compounds of    the invention with a K_(i) value below 5 nM, e.g. compounds Nos.    1-11, 13, 14, 16-18, 20-33, 35, 38, 39, 46 and 48-56.-   2. The K_(i) value of the plasma kallikrein inhibition was likewise    generally <100 nM. The K_(i) value was distinctly less than 100 nM    in particular for compounds with an aromatic carbocyclic system at    R₂ and R₃. Surprisingly, there is a particularly large number of    compounds of the invention with a K_(i) value below 1 nM, e.g.    compounds Nos. 1-3, 5-6, 8-25, 27, 29, 34-36, 39, 40, 49-57, 59-61,    64, 65 and 68-70.-   3. It was possible by incorporating homotyrosine or pyridine and the    corresponding N-oxides as heterocycles in P3 to reduce distinctly    the selectivity for FXa.-   4. It was generally possible to achieve a distinct reduction in the    inhibition of thrombin when R₁ represents a 3-COOH group. A further    reduction in the inhibition of thrombin was achieved when R₄    represents a fluorine atom, especially in ortho position.-   5. A particularly suitable compound has proved to be the compound of    formula (I) with i=0 and without R₄ with the following residues:

Com- pound No. R₁ R₂ p R₃ o 3 3- COOH

3

1

4. Antifibrinolytic and Anticoagulant Properties of Compound No. 3EXPERIMENTAL

In the in vitro studies described below, the antifibrinolytic efficacyof Compound No. 3, aprotinin, and tranexamic acid was compared. Inaddition, the anticoagulant effects of Compound No. 3 were assessedthrough measurement of plasma and whole blood clotting times andthrombin generation.

Aprotinin and tranexamic acid were purchased from SIGMA (Schnelldorf,Germany). Human factor Xa, factor XIa, factor XIIa, thrombin, and plasmakallikrein were purchased from Enzyme Research Laboratories, and humanplasmin from Chromogenix (both via Haemochrom, Essen, Germany). Varioussynthetic peptide chromogenic substrates used for determination ofinhibition constants were obtained from Pentapharm (Basel, Switzerland),Roche (Mannheim, Germany), and Chromogenix: Chromozyme PL for plasmin,S2302 for plasma kallikrein, Pefachrome FXa for factor Xa, Chromozym XIIfor FXIIa, Pefachrom tPA for thrombin, and Pefachrome PCa for factorXIa. Tissue-type plasminogen activator was obtained from BoehringerIngelheim (Ingelheim, Germany). INTEM, EXTEM and STARTEM reagents anddisposables for ROTEM® measurements were obtained from Pentapharm(Munich, Germany).

Venous blood was withdrawn from the antecubital vein from healthyvolunteers after written informed consent in accordance with local andfederal guidelines with approval of the local review board(Ethikkommission, Klinikum Charité, Sauerbruchweg 5, 10117 Berlin). Theblood was mixed with 0.11 mol/L sodium citrate (1:10). For ROTEM®analysis, whole citrated blood was used within 8 h after collection.Platelet-rich plasma (PRP) was prepared from citrated blood bycentrifugation at 330×g for 10 min at room temperature, platelet-poorplasma (PPP) was collected following centrifugation at 1220×g for 12min. The PRP was then adjusted to 3×10⁸ platelets per mL using theautologous PPP. PRP was maintained at room temperature for less than 4 hbefore analysis. The remaining PPP was subjected to a high-speedcentrifugation at 40,000×g for 30 min at 4° C. to remove any particulatematerial and was then stored at −70° C. until use.

Statistics

Statistical analyses were performed using SigmaPlot® 9.0 (SSI, San Jose,Calif.) and SPSS software (SPSS Inc., Chicago, Ill., USA). Data arepresented as mean±standard deviation (SD) or median with 25% and 75%percentile for non-normal distributed measurements.

Differences among groups were assessed by non-parametric Kruskal-Wallistest followed by pairwise post-hoc comparisons using Mann-Whitney-Utest. Assessment of differences between two related samples wasconducted by Wilcoxon signed ranks test. To reduce multiple test issues,Bonferroni correction of p-values was applied within each many-one groupcomparison (several concentrations versus one control) as well in anypairwise post-hoc comparison. A p-value <0.05 was considered to outlinestatistical significance.

(A) Inhibition Constants (K_(i)) of Compound No. 3 and Aprotinin AgainstHuman Serine Proteases

Experimental Design

Inhibition of purified human serine proteases by Compound No. 3 andaprotinin was studied using the established methods described herein inExample 3 (i.e., Stürzebecher et al., 1997). Enzyme kinetic experimentswere carried out in 96-well flat-bottom plates (Brand, Wertheim,Germany) in 50 mM Tris-HCl pH 8.0, 154 mM NaCl in the presence ofdifferent substrate and inhibitor concentrations. Steady-statevelocities of substrate conversion were obtained from progress curvesgenerated by continuous monitoring of the absorbance at 405 nM with amicroplate reader (Multiskan Ascent, Thermo Electron Corporation,Dreieich, Germany). For determination of K_(i) values below 1 nM,measurements were performed in acrylic cuvettes (Brand, Wertheim,Germany) using a UV/VIS spectrophotometer (Specord® M-400, Carl Zeiss,Jena, Germany). Inhibition constants (K_(i)) were calculated fromnon-linear fits of individual data sets to the Michaelis-Menten equationfor competitive inhibitors using an enzyme kinetic analysis software(SIGMA Plot® 9.0 Enzyme Kinetics Module, SSI, San Jose, Calif.). Dixonplot analysis was applied to confirm the competitive inhibition mode.

Results

The results of these enzyme kinetic experiments are summarized inTable 1. Compound No. 3 and aprotinin show comparable inhibition ofplasmin, whereas Compound No. 3 displayed substantially strongerinhibition of plasma kallikrein (2000-fold), FXa (1200-fold) and FXIa(100-fold).

Compound No. 3 Aprotinin K_(i) [nM] K_(i) [nM] Plasmin  2.2 ± 0.2  4.2 ±0.4 Plasma kallikrein  0.019 ± 0.003 38 ± 2 FXa 45 ± 5 55,600 ± 400  FXIa 18 ± 1 1840 ± 40  FXIIa (alpha) 5200 ± 400 5400 ± 100 Thrombin 1700± 200 76,000 ± 2,000(B) Establishment of Antifibrinolytic Potency

Experimental Design

The antifibrinolytic activity of Compound No. 3 in comparison toaprotinin and tranexamic acid was investigated in plasma and whole bloodassays. In both assays, tissue factor was added to initiate rapid clotformation via the extrinsic pathway which produces a clot that remainsstable for several hours under normal conditions. Supplementation ofplasma or whole blood with tissue-type plasminogen activator (t-PA)before stimulation results in activation of endogenous plasminogen andthus fibrinolysis while the initial clot formation is not impaired. Theamount of t-PA added was found to determine lysis time. A finalconcentration of 50 U/mL and 100 U/mL in plasma and whole blood,respectively, was chosen to achieve complete lysis within about 60 min.In the presence of antifibrinolytics, clot lysis is delayed in aconcentration-dependent manner.

a) Plasma Fibrinolysis Assay:

Inhibition of fibrinolysis in plasma was examined using a turbidometricmethod in 96-well flat-bottom plates. The time course of clot formationand lysis, reflected by an initial increase and subsequent decrease inturbidity, was recorded by continuously measuring the optical density(OD) at 405 nm. Similar models have been used widely to study the clotlysis process in human plasma (Kim et al., J. Thromb. Haemost. 5:1250-6, 2007) as well as its inhibition by antifibrinolytic drugs(Sperzel et al., J. Thromb. Haemost. 5: 2113-8, 2007). Frozen and thawedhuman plasma (PPP) was pre-incubated with test compound or vehicle(Owren's Veronal buffer) for 5 min at 37° C. Coagulation and subsequentfibrinolysis was started by adding tissue factor (Innovin®, Dade Behringat 1:9000 final dilution), CaCl₂ (12 mM final concentration) and t-PA(50 U/mL final concentration) simultaneously to the wells. OD at 405 nmwas monitored every 45 s for 180 min at 37° C. with a microplate reader(POLARstar OPTIMA, BMG Labtech, Offenburg, Germany). Fibrinolysis wasquantified as the relative decrease in OD at 45 min after the maximum ODwas reached. Compound No. 3 and aprotinin were each tested atconcentrations of 60, 100, 200, 300, 600, 1000, and 3000 nM, tranexamicacid was tested at concentrations of 600, 1000, 3000, 6000, 10,000,20,000, and 30,000 nM to cover the complete concentration-response foreach compound. These aprotinin concentrations range between 2.8 and 140kallikrein inhibiting units (KIU)/mL based on the conversion factor of7.14 KIU/μg. Concentration-response curves were established by plottingpercentage fibrinolysis versus test compound concentration.

b) Whole Blood Fibrinolysis Assay:

Fibrinolysis in whole blood was studied with rotationalthromboelastometry (Luddington, Clin. Lab. Haematol. 27: 81-90, 2005)using a computerized, multi-channel ROTEM® instrument (Pentapharm,Munich, Germany) (Ganter et al., Anesth. Analg. 106: 1366-75, 2008).Activation of test samples accelerated the measurement process andenhanced reproducibility compared with conventional thromboelastography.To allow observation of fibrinolysis, ROTEM® analysis with tissue factoractivation (EXTEM) was modified through addition of t-PA (Nielsen et al.Blood Coagul. Fibrinolysis 17: 75-81, 2006). Citrated blood waspre-incubated at 37° C. for 5 min with test compound or saline beforetissue factor, CaCl₂ and t-PA (100 U/mL final concentration) were addedto start the reaction. Fibrinolysis was determined by measuring loss ofclot strength with time and was recorded as Ly60 (percentage reductionof the maximum amplitude at 60 min after the onset of clotting). Incontrol samples without inhibitor, clots were lysed completely within 60min, such that Ly60 was above 90%. By plotting Ly60 versus test compoundconcentration, IC₅₀ values for each compound were calculated.

Results

The effect of Compound No. 3, aprotinin, and tranexamic acid on thedynamics of clot formation and lysis in whole blood ROTEM® is depictedin FIGS. 1A-1K. All three agents have equivalent antifibrinolyticefficacy; however, potency differs significantly: Compound No. 3 andaprotinin largely suppress clot lysis at concentrations of 600 and 1000nM, respectively, while tranexamic acid requires concentrations between3000 and 10,000 nM for effective inhibition. Both Compound No. 3 andaprotinin produce a concentration-dependent decrease of Ly60 (FIG. 2A).The concentrations resulting in 50% suppression of clot lysis (IC₅₀,median [25%; 75% percentile]) are 150 [115; 210] nM and 345 [304; 497]nM (corresponding to 16 KIU/mL) for Compound No. 3 and aprotinin,respectively (p<0.001 for comparison of Compound No. 3 vs. aprotinin).Tranexamic acid also reduced clot lysis in a concentration-dependentmanner, although with substantially lower potency (IC₅₀=2750 [1875;3225] nM, p<0.001 vs. Compound No. 3, p=0.002 vs. aprotinin).

Similar results were obtained when human plasma was used instead ofwhole blood as shown in FIG. 2B. Compound No. 3 and aprotinin exhibitcomparable potency on t-PA-induced fibrinolysis in plasma, with IC₅₀values of 315 [135; 506] nM and 327 [280; 537] nM (15 KIU/mL),respectively (p=0.9 for comparison of Compound No. 3 vs. aprotinin).Tranexamic acid exhibits an IC₅₀ of 4225 [3050; 4280] nM (p<0.001 vs.Compound No. 3 and aprotinin), indicating its significantly lowerantifibrinolytic potency.

(C) Assessment of Anticoagulant Potency

Experimental Design

Since Compound No. 3 inhibits multiple proteases of the coagulationsystem, we investigated possible anticoagulant properties usingestablished tests in vitro. Whereas tissue factor is the physiologictrigger of coagulation, both extrinsic and contact-mediated stimulationcontribute to hemostatic activation under conditions like CPB (Boisclairet al. Blood 82: 3350-7, 1993 and Edmunds et al., Ann. Thorac. Surg. 82:2315-22, 2006). We therefore studied the impact of Compound No. 3 andaprotinin on coagulation in plasma and whole blood following bothintrinsic and extrinsic stimulation. Tranexamic acid has no influence onother proteases than plasminogen and, therefore, is not included inthese experiments.

a) Plasma Coagulation Times:

Prothrombin time and activated partial thromboplastin time weredetermined after human plasma (PPP) was supplemented with test compoundsolution or saline using a conventional coagulation analyzer (SysmexCA-560, Dade Behring). Reagents used were Innovin® (extrinsic activatorcontaining tissue factor) for prothrombin time and Actin® FSL (contactactivator containing ellagic acid and phospholipids) for activatedpartial thromboplastin time, both from Dade Behring (Eschborn, Germany).

b) Whole Blood Coagulation Assay:

The influence on whole blood clotting was assayed with rotationalthrombelastometry (Ganter et al., Anesth. Analg. 106: 1366-75, 2008)using ellagic acid (INTEM reagent) as activator of the intrinsic systemor tissue factor (EXTEM reagent) as extrinsic coagulation activator.Following a 5-min pre-incubation with test compounds or saline, citratedhuman blood was subjected to ROTEM® analysis according to themanufacturer's instructions. ROTEM® clotting time (equal to reactiontime, r) and maximum clot strength (equal to maximum amplitude) wereobtained as coagulation parameters.

c) Thrombin Generation Assay:

The impact of Compound No. 3 and aprotinin on thrombin generation wasstudied in PRP using the commercially available Technothrombin® TGA kit(Technoclone, Vienna, Austria). This method allows assessment of thedynamics of thrombin generation, i.e. initiation, propagation, andinactivation phases, including the contribution of platelet function tothe clotting process (Hemker et al., Pathophysiol. Haemost. Thromb. 33:4-15, 2003). PRP was spiked with test compounds at differentconcentrations and pre-warmed to 37° C. in a black 96-well flat-bottomplate (Nunc, Wiesbaden, Germany). Thrombin generation was then initiatedby adding a mixture of activator and fluorogenic thrombin substrate. Twodifferent activators were used; a tissue factor-containing reagentprovided by the manufacturer for extrinsic stimulation, and Actin FSL®(Dade Behring, Eschborn, Germany) at 1:120 final dilution for intrinsicstimulation of thrombin generation. Starting immediately after additionof reagents, fluorescence was recorded every 60 s for 120 min using theBMG POLARstar microplate reader (BMG Labtech, Offenburg, Germany) set at390 nm excitation and 460 nm emission maintaining a temperature of 37°C. Data analysis was performed with the Technothrombin® softwareprovided by Technoclone. A typical thrombin generation curve isgenerated by plotting the first derivative (dF/dt) of the originalfluorescence versus time curve and comparing it to a standard runcontaining known amounts of thrombin in buffer. From these curvesrepresenting the time course of thrombin activity the followingparameters are derived: the lag phase (in min) from time zero until thestart of thrombin generation, peak thrombin level (in nM) and the areaunder the thrombin generation curve (endogenous thrombin potential, innM*min).

Results

In addition to its inhibition of plasma kallikrein, Compound No. 3 alsoaffects factors Xa and XIa. Hence, a significant prolongation of plasmaand whole blood coagulation times was observed in the presence ofCompound No. 3 at antifibrinolytic concentrations ranging from 100 to1000 nM (see FIG. 3). These effects were more pronounced upon intrinsicactivation—reflected in activated partial thromboplastin time and INTEMresults—compared to tissue factor activation—represented by prothrombintime and EXTEM clotting time. Aprotinin had almost no influence onplasma or whole blood coagulation at equivalent concentrations (see FIG.3). A marked prolongation of both activated partial thromboplastin timeand intrinsic ROTEM® clotting times occurred at higher aprotininconcentrations whereas extrinsic coagulation was not affected (data notshown).

Neither Compound No. 3 nor aprotinin impaired clot strength—reflected inROTEM® maximum amplitude—in any of the ROTEM® assays over theconcentration range tested (data not shown).

Compound No. 3 also had a similar effect on thrombin generationfollowing both intrinsic and extrinsic activation that was statisticallysignificant compared to aprotinin as illustrated in FIGS. 3 and 4A-F. Aconcentration-dependent delay in the onset of thrombin generation aswell as a reduction in the peak thrombin level was observed whereasthere was no impact on endogenous thrombin potential in the presence of100 to 1000 nM Compound No. 3.

Discussion of Examples 4A-C

This study demonstrates the efficacy and potency in vitro of thesynthetic, small molecule direct serine protease inhibitor Compound No.3. The findings are summarized as follows: First, Compound No. 3 andaprotinin have almost similar nanomolar potency (2.3 vs. 4.2 nM)regarding inhibition of plasmin enzymatic activity. Second, consistentwith K_(i) data, Compound No. 3 and aprotinin display similar nanomolarpotencies at inhibiting clot lysis in whole blood (IC₅₀ of 150 vs. 345nM) and plasma (IC₅₀ of 315 vs. 327 nM), both drugs being ˜10-fold morepotent than tranexamic acid. Third, Compound No. 3 and aprotinin, butnot tranexamic acid, display anticoagulant properties, with Compound No.3 being more potent than aprotinin as assessed by ROTEM®, global plasmacoagulation tests, and inhibition of thrombin generation. Thus, CompoundNo. 3 is an inhibitor of fibrinolysis that is at least equivalent toaprotinin and more potent compared to tranexamic acid in allinvestigations in vitro so far.

Compound No. 3 offers a number of potential benefits compared toaprotinin; it is a synthetic compound with no risks of transmittinganimal-derived diseases; it has a low molecular weight, so it isunlikely to elicit anaphylactic reactions; finally, due to its shorterhalf-life—terminal half-life is 20 min in rats and dogs—stable plasmaconcentrations may be more easily controlled.

In summary, Compound No. 3 is a small synthetic antifibrinolyticcompound which concentration-dependently inhibits several serineproteases of the hemostatic system. It is not of animal origin and itsprofile is comparable to that of aprotinin with a stronger impact on thecoagulation enzymes factor Xa and plasma kallikrein. Due to its lowmolecular weight, antigenicity is unlikely.

Additional References

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Other Embodiments

The content of each publication, patent, and patent applicationmentioned in the present application is incorporated by reference.Although the invention has been described in details herein andillustrated in the accompanying drawings, it is to be understood thatthe invention is not limited to the embodiments described herein andthat various changes and modifications may be effected without departingfrom the scope or spirit of the invention.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth, and as follows in the scopeof the appended claims.

What is claimed is:
 1. A method of treating or preventing blood loss,said method comprising administering to a subject in need thereof acompound of the formula (I)

or a salt thereof, wherein R₁ is optionally present one or more timesand each R₁ is, independently, hydrogen or COOR₅; R₂ is an optionallysubstituted ring system wherein said ring system is an aromatic ornonaromatic cyclic or bicyclic system comprising 5-13 carbon atoms; R₃is an optionally substituted ring system wherein said ring system isselected from (i) an aromatic cyclic system comprising 5-6 carbon atoms,and (ii) an aromatic heterocycle comprising 3-5 carbon atoms and 1-2nitrogen atoms, a nitrogen oxide, oxygen atom, or sulfur atom; R₄ isoptionally present one or more times and each R₄ is, independently,hydrogen or a halogen; R₅ is hydrogen, a branched or linear lower alkylgroup comprising 1-6 carbon atoms, a branched or linear aminoalkylresidue comprising 1-6 carbon atoms, a halogen or pseudohalogen residue,or a polyethylene glycol residue of the formula (II) or (III):CH₃—O—(CH₂—CH₂—O—)_(n)—CH₂—CH₂—(C═O)—NH—CH₂—  (II)CH₃—O—(CH₂—CH₂—O—)_(n)—CH₂—CH₂—NH—(C═O)—CH₂—CH₂—(C═O)—NH—CH₂—  (III) wherein said polyethylene glycol residue has a molecular weight of from750 Da to 10,000 Da, and n is an integer from 25 to 250; o=1, 2 or 3;p=0, 1, 2, 3 or 4; i=0 or
 1. 2. The method of claim 1, wherein R₁ is aCOOR₅ residue present once and in the meta or para position; R₂ is anoptionally substituted ring system wherein said ring system is phenyl ornapthyl; R₃ is phenyl or phenyl substituted with an alkylamino residuehaving 1-3 carbon atoms; R₄ is hydrogen; R₅ is hydrogen or a branched orlinear lower alkyl group having 1-6 carbon atoms; o is 1 or 2; and i is0.
 3. The method of claim 1, wherein the compound, or the salt thereof,is defined as follows: Compound No. R₁ R₂ p R₃ o i R₄ 1 H

3

1 0 — 2 4-COOH

3

1 0 — 3 3-COOH

3

1 0 — 4 3-COOH

3

1 1 — 5 3-COOH

3

1 0 2-F 6 H

3

1 0 — 7 H

3

1 0 — 8 H

3

1 0 — 9 H

3

1 0 — 10 H

3

1 0 — 11 H

3

1 0 — 12 H

3

1 0 — 13 H

1

1 0 — 14 H

1

1 0 — 15 H

1

1 0 — 16 4-COOH

1

1 0 — 17 H

1

1 0 — 18 3-COOH

1

1 0 — 19 4-COOH

1

1 0 — 20 H

1

1 0 — 21 H

3

1 0 — 22 H

0

1 0 — 23 3-COOH

0

1 0 — 24 H

2

1 0 — 25 3-COOH

2

1 0 — 26 H

2

2 0 — 27 H

2

2 0 — 28 H

3

1 0 — 29 4-COOH

3

1 0 — 30 3-COOH

3

1 0 — 31 H

3

2 0 — 32 H

3

1 0 — 33 3-COOH

3

1 0 — 34 H

3

2 0 — 35 4-COOH

3

2 0 — 36 3-COOH

3

2 0 — 37 H

1

2 0 — 38 H

1

2 0 — 39 H

3

2 0 — 40 3-COOH

3

2 0 — 41 H

3

2 0 — 42 3-COOH

3

2 0 — 43 H

1

2 0 — 44 H

2

2 0 — 45 H

1

2 0 — 47 H

2

2 0  —.


4. The method of claim 1, wherein said blood loss occurs inhyperfibrinolytic conditions.
 5. The method of claim 1, wherein saidcompound, or salt thereof, is administered during a surgical operation.6. The method of claim 5, wherein said surgical operation is a cardiacsurgical procedure or an organ transplant.
 7. The method of claim 6,wherein said cardiac surgical procedure includes cardiopulmonary bypass(CPB).
 8. A method of treating blood loss, said method comprisingadministering to a subject in need thereof a compound having thefollowing structure:

or a salt thereof.
 9. The method of claim 8, wherein said blood lossoccurs in hyperfibrinolytic conditions.
 10. The method of claim 8,wherein said compound, or salt thereof, is administered during asurgical operation.
 11. The method of claim 10, wherein said surgicaloperation is a cardiac surgical procedure or an organ transplant. 12.The method of claim 11, wherein said cardiac surgical procedure includescardiopulmonary bypass (CPB).
 13. A method of preventing blood loss,said method comprising administering to a subject in need thereof acompound having the following structure:

or a salt thereof.
 14. The method of claim 13, wherein said blood lossoccurs in hyperfibrinolytic conditions.
 15. The method of claim 13,wherein said compound, or salt thereof, is administered during asurgical operation.
 16. The method of claim 15, wherein said surgicaloperation is a cardiac surgical procedure or an organ transplant. 17.The method of claim 16, wherein said cardiac surgical procedure includescardiopulmonary bypass (CPB).
 18. A method of administering to a subjectin need thereof a compound having the following structure:

or a salt thereof, wherein said patient is undergoing blood loss. 19.The method of claim 18, wherein said compound, or salt thereof, isadministered to a subject undergoing a surgical operation.
 20. Themethod of claim 19, wherein said surgical operation is a cardiacsurgical procedure or an organ transplant.
 21. The method of claim 20,wherein said cardiac surgical procedure includes cardiopulmonary bypass(CPB).